The long term goal of the proposed research is to isolate and characterize a cytoplasmic antigen that stimulates protective immunity to an intracellular parasite Toxoplasma gondii. We will further define the specificity of the antigen purified from an affinity chromatography column with the aid of western blotting and enzyme linked immunosorbent assays. The molecular weight of the antigen will be determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments will be performed to determine whether the purified antigen is a protein or a glycoprotein. Chemical and proteolytic cleavage of the purified antigen will be carried out to define the location of the epitopes recognized by the monoclonal antibody. We will clone the gene coding for this protein and develop methodology to obtain large quantities of this antigen. Studies will be conducted to define the mechanisms by which purified antigen, antigenic fragments obtained as a result of proteolytic and chemical cleavage as well as the antigen produced by recombinant DNA methods induces a protective immune response. The ability of these antigenic preparations to prime spleen cells to secrete interferon, activate macrophages, produce different classes of immunoglobulins and affect the number of cysts formed will be examined. We hope the proposed research will lead to a better understanding of the nature of the antigenic structure of Toxoplasma and the mechanisms by which well characterized antigens stimulate an immune response beneficial to the host. Our gene cloning experiments will not only allow us to produce large quantities of the antigen but will also open the doors for future studies concerned with the regulation of genes coding for the antigens of Toxoplasma gondii.